PROJECT SUMMARY Influenza virus is a major health burden worldwide, resulting in billions of dollars in medical costs and up to 600,00 deaths annually. Thus, a major challenge of pulmonary vaccinology is to develop an approach that will provide long-lasting and durable immunity in the lung. Seasonal influenza infection is driven largely by antigenic shift to avoid established antibody responses, limiting the efficacy of current influenza vaccinations designed to generate humoral immunity to the hemagglutinin protein. One approach to this problem is the development of vaccines designed to promote the generation of influenza-specific, lung-resident T cell memory. In animal models and human studies, memory T cells have been shown to significantly reduce viral loads after influenza challenge, leading to faster viral clearance, decreased transmission, and milder or sub- clinical symptoms. Lung tissue-resident memory T cells (lung TRM) have been found to be critical for this protective cellular response to influenza, but lung TRM numbers gradually decline over time. Despite thie importance for pulmonary immunity, we still have only a basic understanding of the cellular and molecular mechanisms that control their generation and long-term maintenance, nor have we identified the optimal vaccination strategies to induce durable lung TRM. Furthermore, the influence on unique microenvironments of the lung interstitium and lung airways on TRM biology is poorly understood. This proposal seeks to develop a program in lung TRM biology centered on three related themes: (i) investigating the molecular programming of TRM that enables their generation following infection and vaccination; (ii) defining the cell- and tissue-intrinsic mechanisms that drive the gradual loss of lung TRM and testing the ability of novel vaccinations strategies to improve lung TRM longevity; and (iii) determining the transcriptional and epigenetic programming of influenza- specific lung TRM at the bulk and single cell level using a biorepository of human lung samples. The overall goal of this project is to understand the mechanisms driving the initial generation and subsequent decline of influenza-specific TRM from the lung interstitium and airways. The knowledge gained in this study will provide a deep understanding of the mechanisms that regulate lung TRM biology to better inform future vaccine design against respiratory pathogens.